RESUMEN
Nevoid basal cell carcinoma syndrome (NBCCS), or Gorlin's syndrome, is an autosomal dominant disorder that predisposes to developmental defects and various forms of cancer. PTCH was recently proposed as a candidate gene for NBCCS due to its frequent mutation in basal cell carcinomas, the cancer most often associated with this syndrome. Another NBCCS-associated cancer is medulloblastoma, a common central nervous system tumor in children. Most medulloblastomas, however, occur without indication of an inherited predisposition. We have examined 24 sporadic medulloblastomas for loss of heterozygosity (LOH) at loci flanking as well as within PTCH. In cases with LOH, single-strand conformational polymorphism and sequencing analysis were performed to determine the status of the remaining PTCH allele. Microsatellite analysis indicated LOH of PTCH in 5 of 24 tumors, and in three of these cases a mutation of the remaining allele was identified. Two of the mutations were duplication insertions, and the third consisted of a single base deletion. It is interesting that all three mutations occur in exon 17 of the PTCH gene. These data suggest that inactivation of PTCH function is involved in the development of at least a subset of sporadic medulloblastomas.
Asunto(s)
Meduloblastoma/genética , Proteínas de la Membrana/genética , Cromosomas Humanos Par 9 , Heterocigoto , Humanos , Repeticiones de Microsatélite , Receptores Patched , Receptor Patched-1 , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo Conformacional Retorcido-Simple , Receptores de Superficie Celular , Eliminación de SecuenciaRESUMEN
Loss of heterozygosity (LOH) from chromosome 10 is a hallmark of glioblastoma, the most malignant (grade IV) form of glioma. A candidate tumor suppressor gene, PTEN/MMAC1, that may be targeted for deletion in association with chromosome 10 LOH has recently been identified. Here we have investigated 63 glioblastomas for PTEN/MMAC1 alterations and identified DNA sequence changes that would affect the encoded protein in 17 (27%) tumors. Microsatellite analyses of normal-tumor DNA pairs were performed on 14 of these cases and revealed LOH at locations flanking and/or near PTEN/MMAC1 in all but 1 instance, suggesting that deletion of the remaining wild-type allele had occurred in the large majority of tumors with PTEN/MMAC1 mutations. Competitive PCR assays were developed to address the possible occurrence of PTEN/MMAC1 homozygous deletions in glioblastomas, and this analysis identified three samples having loss of both PTEN/MMAC1 alleles. EGFR amplification was determined to occur at similar frequencies among cases with or without PTEN/MMAC1 homozygous deletions or mutations, suggesting that a growth-promoting effect resulting from amplification-associated increases in epidermal growth factor receptor signaling is not necessarily dependent on the inactivation of PTEN/MMAC1.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 10 , Receptores ErbB/genética , Glioblastoma/genética , Mutación , Monoéster Fosfórico Hidrolasas , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/genética , Proteínas Supresoras de Tumor , Empalme Alternativo , Secuencia de Aminoácidos , Mapeo Cromosómico , ADN/sangre , ADN de Neoplasias/química , Receptores ErbB/biosíntesis , Receptores ErbB/química , Exones , Mutación del Sistema de Lectura , Glioblastoma/enzimología , Glioblastoma/cirugía , Humanos , Repeticiones de Microsatélite , Datos de Secuencia Molecular , Fosfohidrolasa PTEN , Mutación Puntual , Reacción en Cadena de la Polimerasa , Proteínas Tirosina Fosfatasas/química , Eliminación de SecuenciaRESUMEN
Joint predisposition to malignant melanoma and nervous system tumors (NSTs) is a puzzle. Several melanoma susceptibility genes have been identified, including p16, a clustered tumor suppressor. However, the molecular bases of inherited proclivity to NSTs in the absence of a recognizable genetic syndrome are unknown. We analyzed two families with joint proneness to melanoma and NSTs in view of genetic linkage and identification of the causal molecular lesions. Highly informative linkage markers were used for segregation analyses of the predisposition alleles in the two pedigrees. Characterization of the molecular lesions required hemizygosity mapping based on microsatellite markers physically mapped to contigs of the 9p21 region and a Southern blot approach using several PCR-generated probes. Both families were found to be allelic and linked to p16 markers. In the family segregating the melanoma/NST syndrome, a large germ-line deletion ablated the whole p16, p19, and p15 gene cluster (or INK4 locus), whereas a more circumscribed molecular lesion disrupting p16 and p19 but leaving p15 unaltered segregated with the melanoma-astrocytoma syndrome (MIM 155755). Our results suggest that multiple cancer susceptibility in these two families ensues from contiguous tumor suppressor gene deletion. Indeed, known phenotypes associated with germ-line p16 mutations and an apparent correlation between the deletion span and tumor spectrum in the two families suggest a new model of cancer pathogenesis based on the inactivation of contiguous tumor suppressor genes, an alternative to the established pleiotropic effects of single-gene disruption.
Asunto(s)
Proteínas de Ciclo Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Eliminación de Gen , Melanoma/genética , Neoplasias Primarias Secundarias/genética , Síndromes Neoplásicos Hereditarios/genética , Neoplasias del Sistema Nervioso/genética , Proteínas Supresoras de Tumor , Adulto , Anciano , Alelos , Proteínas Portadoras/genética , Cromosomas Humanos Par 9 , Inhibidor p15 de las Quinasas Dependientes de la Ciclina , Inhibidor p19 de las Quinasas Dependientes de la Ciclina , Femenino , Genes p16 , Predisposición Genética a la Enfermedad , Humanos , Masculino , Repeticiones de Microsatélite , Linaje , Análisis de Secuencia de ADNRESUMEN
Allelic alterations of chromosomes 1 and 19 are frequent events in human diffuse gliomas and have recently proven to be strong predictors of chemotherapeutic response and prolonged survival in oligodendrogliomas (Cairncross et al., 1998; Smith et al., submitted). Using 115 human diffuse gliomas, we localized regions of common allelic loss on chromosomes 1 and 19 and assessed the association of these deletion intervals with glioma histological subtypes. Further, we evaluated the capacity of multiple modalities to detect these alterations, including loss of heterozygosity (LOH), fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH). The correlation coefficients for detection of 1p and 19q alterations, respectively, between modalities were: 0.98 and 0.87 for LOH and FISH, 0.79 and 0.60 for LOH and CGH, and 0.79 and 0.53 for FISH and CGH. Minimal deletion regions were defined on 19q13.3 (D19S412-D19S596) and 1p (D1S468-D1S1612). Loss of the 1p36 region was found in 18% of astrocytomas (10/55) and in 73% (24/33) of oligodendrogliomas (P < 0.0001), and loss of the 19q13.3 region was found in 38% (21/55) of astrocytomas and 73% (24/33) of oligodendrogliomas (P = 0.0017). Loss of both regions was found in 11% (6/55) of astrocytomas and in 64% (21/33) of oligodendrogliomas (P < 0.0001). All gliomas with LOH on either 1p or 19q demonstrated loss of the corresponding FISH probe, 1p36 or 19q13.3, suggesting not only locations of putative tumor suppressor genes, but also a simple assay for assessment of 1p and 19q alterations as diagnostic and prognostic markers.
Asunto(s)
Neoplasias Encefálicas/genética , Cromosomas Humanos Par 19/genética , Cromosomas Humanos Par 1/genética , Glioma/genética , Eliminación de Secuencia , Astrocitoma/genética , Astrocitoma/patología , Neoplasias Encefálicas/patología , Cromosomas Humanos Par 1/ultraestructura , Cromosomas Humanos Par 19/ultraestructura , Glioma/clasificación , Glioma/patología , Humanos , Hibridación Fluorescente in Situ , Pérdida de Heterocigocidad , Oligodendroglioma/genética , Oligodendroglioma/patologíaRESUMEN
The transfectivity of anthramycin (Atm)-modified phi X174 replicative form (RF) DNA in Escherichia coli is lower in uvrA and uvrB mutant cells but much higher in uvrC mutant cells compared to wild-type cells. Pretreatment of the Atm-modified phage DNA with purified UVRA and UVRB significantly increases the transfectivity of the DNA in uvrA or uvrB mutant cells. This pretreatment greatly reduces the UVRABC nuclease-sensitive sites (UNSS) and Atm-induced absorbance at 343 nm in the Atm-modified DNA without producing apurinic sites. The reduction of UNSS is proportional to the concentrations of UVRA and UVRB and the enzyme-DNA incubation time and requires ATP. We conclude that there are two different mechanisms for repairing Atm-N2 guanine adducts by UVR proteins: (1) UVRA and UVRB bind to the Atm-N2 guanine double-stranded DNA region and consequently release the Atm from the adducted guanine; (2) UVRABC makes an incision at both sides of the Atm-DNA adduct. The latter mechanism produces potentially lethal double-strand DNA breaks in Atm-modified phi X174 RF DNA in vitro.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Antramicina/química , Proteínas Bacterianas/metabolismo , Daño del ADN , ADN Helicasas , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli , Ácido Apurínico/química , ADN Bacteriano/química , ADN Viral/química , Endodesoxirribonucleasas/metabolismo , Mutación , Espectrofotometría Ultravioleta , TransfecciónRESUMEN
The purpose of this study was to examine the incidence of gene amplification in patients with primary (de novo) and secondary high-grade gliomas (gliomas evolving from lower grade malignancies) and to assess its prognostic significance. A total of 186 prospectively collected frozen surgical specimens were analyzed. Extracted DNA was examined by Southern blot using probes corresponding to the EGFR, CDK4, MDM2, n-MYC, CYCD1, PDGFR-alpha, MET, c-MYC oncogenes. Complete clinical data regarding age, sex, tumor size, extent of surgical resection, postoperative therapy and patient survival were collected. We showed that EGFR followed by CDK4 were the most frequent oncogene amplifications. Oncogene amplification events were significantly more frequent in grade 4 than in grade 3 astrocytomas, mixed gliomas or oligodendrogliomas (P<0.001). With respect to EGFR, there was a significant difference in the frequency of amplification between primary and secondary gliomas (P=0.001); however, no difference in the amplification frequency of the other oncogenes was observed. There was no apparent correlation between the occurrence of gene amplification and patient survival, possibly because the genes amplified in human gliomas are part of larger signaling pathways.
Asunto(s)
Neoplasias Encefálicas/genética , Glioma/genética , Oncogenes/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Encefálicas/patología , Niño , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Femenino , Amplificación de Genes , Glioma/patología , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Pronóstico , Modelos de Riesgos Proporcionales , Análisis de SupervivenciaRESUMEN
Capping of concanavalin A (Con A) and anti-Leu-8 (L8) receptors on human peripheral blood mononuclear (PBM) membranes was studied utilizing fluorochrome-conjugated ligands and flow cytometric analysis. Histogram profiles of fluorescent intensities consistently revealed a time-dependent decrease in numbers of brightly fluorescing events concurrent with an increase in numbers of dimly fluorescing events when capping occurs. Differences in fluorescence profiles were detectable by flow cytometric analysis as early as 5 min after capping conditions were initiated. A pronounced defect in receptor capping of PBM cells from AIDS patients was observed. This technique represents a rapid and reproducible means for detecting early changes in cell membrane receptor mobility.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/diagnóstico , Moléculas de Adhesión Celular/metabolismo , Linfocitos/metabolismo , Agregación de Receptores , Receptores de Concanavalina A/metabolismo , Membrana Celular/metabolismo , Citometría de Flujo , Humanos , Selectina L , Microscopía FluorescenteRESUMEN
An anesthetic dose of 50 mg/kg (i.p.) sodium pentobarbital caused a 61% increase in blood glucose levels in mice. Nicotine (2.5 mg/kg) given intraperitoneally 15 min prior to the sodium pentobarbital treatment further increased hyperglycemia by 29% over pentobarbital alone and 90% higher than the control. Mecamylamine (0.5 mg/kg) given intraperitoneally 15 min prior to nicotine resulted in blood glucose concentrations near the control value. Atropine (2 mg/kg) administered intraperitoneally did not prevent the hyperglycemia induced by nicotine and pentobarbital. No significant correlation was observed between the sleep time and the blood glucose of the unconscious or awake mice. However, a significant correlation was observed between the blood glucose concentration of the sleeping and awake mice. Blood glucose levels were always higher when the neuronal activity was depressed and were lower when the neuronal activity was increased.
Asunto(s)
Atropina/farmacología , Hiperglucemia/inducido químicamente , Mecamilamina/farmacología , Nicotina/antagonistas & inhibidores , Pentobarbital/farmacología , Sueño/efectos de los fármacos , Animales , Glucemia/metabolismo , Sinergismo Farmacológico , Hipnosis Anestésica , Masculino , Ratones , Ratones Endogámicos ICR , Nicotina/farmacologíaRESUMEN
Nicotine potentiates, in a dose dependent manner, the sleep time induced by sodium pentobarbital but not by ethanol. Mecamylamine, a nicotinic receptor antagonist, blocked the nicotine induced increase in sleep time. Atropine itself reduced sleep time but did not change the nicotine effect. It is hypothesized that the central and the peripheral nicotinic receptors play an important role in potentiating sodium pentobarbital induced sleep time.
Asunto(s)
Etanol/farmacología , Nicotina/farmacología , Pentobarbital/farmacología , Sueño/efectos de los fármacos , Animales , Atropina/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Mecamilamina/farmacología , Ratones , Ratones Endogámicos , Receptores Nicotínicos/efectos de los fármacos , Factores de TiempoRESUMEN
A chemically synthesized peptide corresponding to the amino acid sequence 503-532 of gp160 of human immunodeficiency virus (HIV) was used to generate monoclonal antibodies reactive with the env glycoprotein gp120. One monoclonal antibody, 120-1, was isolated that reacted with the peptide and with HIV antigen(s). Western blot analysis showed reactivity with two bands of 120 kDa and 88 kDa. 120-1 reacted in indirect immunofluorescence with 15-20% of infected human T cell line A3.01 as early as 4 days post in vitro infection, 2-3 days prior to detectable reverse transcriptase activity in supernatant fluids.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , VIH/inmunología , Proteínas de los Retroviridae/inmunología , Secuencia de Aminoácidos , Antígenos Virales/inmunología , Técnica del Anticuerpo Fluorescente , Anticuerpos Anti-VIH , Antígenos VIH , Proteína gp120 de Envoltorio del VIH , Humanos , Inmunoglobulina M/inmunología , Péptidos/síntesis química , Péptidos/inmunología , Proteínas de los Retroviridae/análisis , Linfocitos T/análisisRESUMEN
Advanced-stage epithelial ovarian cancers (EOC) from 114 patients were assessed for loss of heterozygosity (LOH or allelic imbalance) at several tumor suppressor gene loci as an initial step in identifying gene alterations important to the development of these tumors. The highest frequency of loss, 84% (86 of 102 cases), was observed for markers mapping near or within BRCA1; other significant frequencies of LOH were observed for loci mapping near or within CDKN2A/CDKN2B (56%), BRCA2 (61%), RB1 (67%), or TP53 (73%). No instance of TP53 LOH was observed without simultaneous allelic imbalance at the BRCA1 region (P = 0.0005). LOH of CDKN2 without loss near the BRCA1 region was seen in only 2 of 75 cases (P < 0.0001), and RB1 LOH without BRCA1 loss occurred in only 1 of 35 tumors (P = 0.0703). These data suggest that LOH of BRCA1, or a closely linked locus, precedes the loss of CDKN2, TP53, and RB1, and imply that inactivation of a tumor suppressor gene in this region is an important early step in the development of these tumors.
Asunto(s)
Biomarcadores de Tumor/genética , Proteínas de Ciclo Celular/genética , Genes BRCA1/genética , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Ligamiento Genético , Humanos , Pérdida de Heterocigocidad , Persona de Mediana EdadRESUMEN
p73, a protein having substantial structural and functional similarity to p53, has recently been identified and demonstrated to be a potential tumor suppressor. Its location on human chromosome 1p36.33 implicates p73 as a candidate for neuroblastoma. Like neuroblastoma, oligodendrogliomas also show a high frequency of deletions in chromosome 1p36.3. To determine whether p73 is a potential tumor suppressor gene involved in the development of oligodendrogliomas, we performed mutation analysis of p73 in oligodendrogliomas with chromosome 1 p-arm deletions. We first determined the genomic organization and the intron-exon boundary sequences of the p73 gene by long PCR, vectorette PCR, and Southern hybridization. This gene spans about 65 kb with a large first intron. Primer pairs for the amplification of each of the 13 p73 encoding exons were designed in corresponding introns. The amplicons were then analyzed using the denaturing high-performance liquid chromatography system for mutations in the p73 gene. Twenty oligodendroglioma samples with 1p36.3 deletions were screened, but no mutations were detected except for several polymorphisms. It is thus clear that p73 is not a candidate gene for oligodendroglioma despite its location in the frequently deleted 1p36.3 region.